SUMMARY/ABSTRACT Neuroblastoma (NB) remains a leading cause of childhood cancer morbidity and mortality. We discovered heritable activating mutations in the ALK oncogene, and that these same mutations are frequently somatically acquired during high-risk NB tumorigenesis . Our work established ALK as a tractable molecular target in NB and provided the rationale for the clinical development of ALK inhibition therapy. This second competitive renewal builds on six major discoveries over the last decade: 1) we showed that the majority of activating ALK mutations are not sensitive to first generation drugs such as crizotinib; 2) we demonstrated that chemotherapy could sensitize ALK mutant NBs to crizotinib, leading to an ongoing Phase 3 trial; 3) we identified lorlatinib as the only ALK inhibitor that is effective against all activating mutations and rapidly opened a phase 1 clinical trial that is showing significant anti-tumor activity while still in dose escalation; 4) we developed and implemented a clinical trial matching genomic aberrations in tumor cells at the time of relapse to rationally designed combinations of molecularly targeted agents (e.g. ceritinib + ribociclib for patients with ALK-driven NB) that have shown synergistic activity in our lab; 5) we showed that the superior activity of lorlatinib is mediated through inhibition of G2/M kinases; and 6) we showed that ALK is abundantly expressed on the cell surface of the vast majority of NBs and other pediatric malignancies, and have developed immunotherapeutic strategies since ALK is not expressed on normal tissues. Thus, the long-term goal of this new research proposal is to improve outcomes for children with NB by leveraging the momentum above into rational new combinatorial and immunotherapeutic treatment strategies. The objective here is to identify mechanisms regulating adaptive responses to targeted ALK inhibition with lorlatinib and to develop therapeutic strategies aimed at targeting ALK in the plasma membrane. Our central hypotheses are that i) ALK-driven NBs adapt to and survive therapy with lorlatinib via mechanisms that can be therapeutically targeted; and ii) immunotherapeutic targeting of native ALK is of biologic relevance to the majority of patients with NB and to identifiable subsets of other childhood tumors. We will test our central hypothesis in two specific aims: 1) Elucidate mechanisms of adaptive resistance to ALK-kinase inhibition with lorlatinib to identify optimal combination strategies that will result in improved and sustained therapeutic efficacy; 2) Develop highly specific antibody-based approaches to target cell surface ALK on NBs and other ALK-expressing pediatric cancers. We consider this proposal significant because it will result in a new mechanism-based therapeutic strategy that will address the major unmet need that, despite unprecedented discoveries in defining the basic mechanisms of NB tumorigenesis, this knowledge has not yet translated into significantly improved outcomes.